Human granulocytic anaplasmosis (HGA) is an emerging disease caused by the obligate intracellular bacterium Anaplasma phagocytophilum (Ap), and numbers of new human cases are increasing each year. In North America, Ap is transmitted to humans and animals by the ticks Ixodes scapularis and Ixodes pacificus, with I. scapularis responsible for the vast majority of human cases. Impressive strides have been made in understanding epidemiological, clinical, physiological, and molecular aspects of Ap's interaction with mammals, whereas little progress has been made in understanding the intimate relationship between this bacterium and its vector tick at the cellular level. One significant limitation has been the difficulty in localizing Ap within the tick. We have adapted new techniques that allow us to precisely locate individual bacteria within a tick by labeling unique bacterial RNA sequences in fixed sections of whole ticks, making it possible to visualize Ap-infected tissues and cells in the context of intact tick anatomy, and to distinguish them from other bacteria, including pathogens and endosymbionts. In addition, we have generated a library of single insertion Ap mutants in which there are numerous hits to genes only transcribed during infection of tick cells; these cloned mutants will allow us, in future studies, to identify p genes associated with each stage of the tick colonization process, from acquisition to salivary gland infection. The time is now right to proceed with a careful analysis of Ap colonization of its principal vector, I. scapularis. The main goals of this study are twofold. First, we will analyze te spread of Ap through tick tissues, using confocal microscopy to visualize and quantify Ap in I. scapularis nymphs and adults infected as larvae as these are the life stages involved in transmission. To do so, we will examine histological sections of engorged larvae, unfed nymphs, and engorged nymphs, unfed and partially fed adult ticks using an Ap-specific fluorescent RNA probe. Measurable characteristics of Ap infection such as mean morula (an intracellular inclusion containing bacteria) diameter and proportion of occupied cellular cytoplasm will be quantified for each infected organ type at the various life stages of the tick. Quantitative PCR will be used to estimate Ap load. Second, we will investigate the mechanisms of bacterial trafficking between cells within the body of the tick. Dissemination via hemolymph plasma, transport by viably infected hemocytes, and direct cell-to-cell contact will be examined. Ap transformed to express green-fluorescent protein will be imaged live to determine if it localizes to the destructive lysosomal compartments of phagocytic hemocytes or replicates to generate morulae in the cytoplasm. Further viability tests will be performed in vitro with hemocytes and hemolymph plasma to determine if either component, separately, is capable of infecting cultured cells. To test direct contact infection, Ap-infected organs will be dissected from I. scapularis tiks and placed in contact with cultured cells in the presence of antibiotic-containing media restricting extracellular viability.